Electron-dense material (EDM) appears at the parasite plasma membrane with trophozoites of several strains of Plasmodium falciparum cultured in vitro. The EDM is also seen associated with unit membrane-bounded Maurer's clefts in K+ P. falciparum-infected erythrocytes. The cytoplasmic clefts lack the EDM with K- parasites. Some EDM have the same density and appearance as the material located under knobs at the erythrocyte membrane. The EDM at the parasite plasma membrane is absent with schizonts when expression of new knobs at the erythrocyte membrane appears to have ceased. This electron microscopic study suggests that the parasite-derived EDM is transported from the parasite plasmalemma to the erythrocyte membrane via Maurer's clefts in the erythrocyte cytoplasm.
Freshly prepared spleen cells from concanavalin A stimulated mice incorporate [3H]thymidine into DNA which can be recovered in detergent-soluble (NP40) and detergent-insoluble forms. The presence of detergent-soluble forms occurs despite the fact that the cells are lysed at 4 degrees C in the presence or absence of 25 mM ethylenediaminetetraacetic acid. After a 2-h pulse with [3H]thymidine, the detergent-soluble fraction contains about 1-3% of the total cellular DNA but 25% of the total labeled high molecular weight material. Since the specific activity of the extensively purified DNA from the detergent-soluble fraction is considerably higher than that of chromosomal DNA, it meets the criteria for being metabolically active. We propose the name "MADS" DNA for metabolically active detergent-soluble DNA. MADS DNA has a density of 1.699 g/mL on cesium chloride gradients and a slightly higher G + C content than chromosomal DNA as determined by high-pressure liquid chromatography. Electrophoresis using native or denaturing agarose gels resolves MADS DNA into discreet sizes between 200 and 4500 base pairs. Nuclease S-1 treatment of native MADS DNA does not alter the size distribution as resolved by means of gel electrophoresis under denaturing conditions. Therefore, MADS DNA is not a collection of single-stranded Okazaki fragments. Southern blot analysis reveals that mitochondrial DNA is a minor component of higher molecular weights above the bulk of the DNA visualized either by staining with ethidium bromide or by incorporation of [3H]thymidine. Inhibitors of ribonucleotide reductase or DNA polymerase alpha inhibit incorporation of [3H]thymidine into MADS DNA, and hence chromosomal DNA synthesis is required for MADS DNA production. Since Southern blot analysis also reveals homology of larger fragments with the 32P-labeled 200 base pair fragment, the presence of repetitive sequences is suggested.
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